Copper is an essential nutrient. However, despite its importance to human nutrition, little is known about the mechanisms regulating copper homeostasis in mammalian cells. Copper is required by several important enzymes, however, it is also toxic when present in excess concentrations. Thus, copper homeostasis mechanisms must supply sufficient copper to meet cellular needs, while preventing the over-accumulation of this nutrient. Copper uptake in mammalian cells occurs via the membrane-spanning protein, hCtr1. Currently, we have limited knowledge of whether hCtr1-mediated copper uptake is regulated in response to varying copper availability. In contrast, we have a more extensive knowledge of how copper export is regulated in mammalian cells. Two copper ATPases, ATP7A and ATP7B, which are normally located in the trans-Golgi network, are stimulated to relocate to cytoplasmic vesicles or the plasma membrane by elevated copper to facilitate copper efflux from the cytoplasm. In our preliminary studies, we show that the location of the hCtr1 protein is regulated by copper concentrations. Elevated copper stimulates the rapid endocytosis of hCtr1 from the plasma membrane, and this is associated with degradation of the transporter. We hypothesize that this process is likely to be the principle means by which high affinity copper uptake is regulated in mammalian cells. However, the underlying molecular mechanisms and signals involved have not yet been defined. Our long-term goal is to understand the molecular basis for regulating hCtr1-dependent copper uptake. To achieve this overall goal, we propose the following specific aims: 1.To define the intracellular pathway for copper-stimulated endocytosis and degradation of hCtr1. 2. To identify amino acids within hCtr1 important for copper uptake, copper-induced endocytosis and degradation. 3. To assess whether the localization, endocytosis and degradation of hCtr1 is responsive to intracellular copper levels. 4. To determine whether the hCtr1 protein undergoes copper-stimulated endocytosis and degradation in a range of cell types. Our research will contribute greatly to understanding how cells sense and respond to changes in copper availability. The implication of copper in Alzheimer's disease, prion diseases, and several genetic disorders, suggests the study of hCtr1 may have far-reaching implications for the improvement of human health.